Chromogenic substrates of sialidase and methods of making and using the same

ABSTRACT

The subject invention discloses materials and methods for the design, synthesis, and biochemical evaluation of chromogenic substrate compounds for sialidases of bacterial, viral, protozoa, and vertebrate (including humans) origin. In particular, this invention provides a novel class of effective compounds as chromogenic substrates of these sialidases which yield chromogenic products after reactions catalyzed by sialidase take place. Also provided are methods of making these substrate compounds, methods of diagnosis and prognosis of sialidase related diseases using these substrate compounds.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 10/328,790,filed Dec. 24, 2002, which is a divisional of application Ser. No.09/651,622, filed Aug. 30, 2000, now U.S. Pat. No. 6,512,100 B1 which isa continuation of application Ser. No. 08/958,356, filed Oct. 27, 1997,now abandoned. This application is also a divisional of application Ser.No. 08/958,356, filed Oct. 27, 1997, now abandoned. Each of theseapplications is hereby incorporated by reference in their entireties,including any figures, tables, and drawings.

The research related to this invention is in part supported by acontract from the University of Alabama at Birmingham as a grant fromthe US Defense Advanced Research Projects Agency, grant number MDA972-97-K-0002.

FIELD OF THE INVENTION

The current invention relates to the design, synthesis, and biochemicalevaluation of chromogenic substrate compounds for sialidases ofbacterial, viral, protozoa, and vertebrate (including humans) origin. Inparticular, this invention provides a novel class of effective compoundsas chromogenic substrates of these sialidases which yield chromogenicproducts after reactions catalyzed by sialidase take place. Alsoprovided are methods of making these substrate compounds, methods ofdiagnosis and prognosis of sialidase related diseases using thesesubstrate compounds.

BACKGROUND OF THE INVENTION

Sialidase (EC, 3.2.1.18, also known as neuraminidase, acylneuraminylhydrolase) is a protein enzyme produced by many organisms such asbacteria, viruses, protozoa, and vertebrates including humans (Hirst, G.K. [1941] Science 94:22-23). This class of enzymes catalyzes thehydrolysis of a terminal sialic acid which is linked to oligosaccharidesthrough an O-glycosidic bond. Crystal structure of sialidases showedthat the enzyme has a highly conserved active site centered in apropeller like β-sheet twirl (Crennell, S. J. et al. [1993] Proc. Natl.Acad. Sci. USA 90:9852-9856).

Sialidases perform many critical biological functions. In bacteria,sialidase helps bacterial adhesion to tissues, and provides additionalnutritional sources (Crennell, S. et al. [1994] Structure 2(6):535-544).In viruses, it helps the release of progeny viruses (Liu, C. et al.[1995] J. Virol. 69:1099-1106). In a parasite, Trypanosoma cruzi, asialidase (also known as trans-sialidase) removes sialic acids frominfected cells and decorates its own surface with these sialic acids. Inhumans, sialidases are involved in protein digestion, immune responses,and cell proliferation. Abnormal production of sialidases may lead toserious human diseases such as sialidosis or increased Pseudomonasaeruginosa infection in cystic fibrosis patients.

Since sialidases are associated with many diseases, a color-producingsubstrate of sialidase would be an excellent diagnostic or prognosticreagent for sialidase-related diseases. For instance, sialidase level iselevated in bacterial vaginosis (Briselden, A. M. et al. [1992] J. Clin.Microbiol. 30:663-666). Measurement of sialidase level in the vaginalsamples could be used to diagnose bacterial vaginosis. In periodontaldisease caused by bacterial infection, it has been shown that presenceof sialidase increases the colonization of harmful bacteria (Liljemark,W. F. et al. [1989] Caries Res. 23:141-145). The cell invasion form ofT. cruzi, Trypomastigote, expresses high levels of trans-sialidaseactivity; therefore, measurement of trans-sialidase level could be usedfor diagnosis of T. cruzi infection and for monitoring disease progress(Cross, G. A., G. B. Takle [1993] Annu. Rev. Microbiol. 47:385-411). Incystic fibrosis patients, Pseudomonas aeruginosa infection is one of theleading causes of death. Sialidase was shown to be involved in thedisease progress (Cacalano, G. et al. [1992] J. Clin. Invest.89:1866-1874). Sialidase is also related to the regulation of cellproliferation (Bratosin, D. et al. [1995] Glycoconj. J. 12:258-267), theclearance of plasma proteins (Bonten, E. et al. [1996] Genes & Devel.10:3156-3169), and the catabolism of gangliosides and glycoproteins(Gornati, R. et al. [1997] Mol. Cell Biochem. 166:117-124).

Currently, there is available a synthetic substrate of sialidase,4-methylumbelliferyl-B-acetyl-neuraminic acid (4-MUN) (Lentz, M. R., R.G. Webster, G. M. Air [1987] Biochemistry 26:5351-5358), which producesa product with characteristic fluorescence spectrum upon hydrolysis.This change of fluorescence spectrum can only be measured with aspecialized instrument (fluorospectrometer). The substrate compounds ofthe current invention produce a visible color change upon hydrolysis,which is highly advantageous in medical diagnostic applications.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the current invention relates to the design andsynthesis of novel chromogenic substrate compounds for sialidases. Inanother embodiment, the subject invention pertains to the use of thenovel chromogenic substrates in assays for the detection of sialidases.The sialidases which are detected using the procedures and compounds ofthe subject invention are of bacterial, viral, protozoa, and vertebrate(including human) origin. In a specific embodiment, the subjectinvention provides a novel class of compounds which are useful aschromogenic substrates of sialidases.

In one embodiment, the present invention provides chromogenic sialidasesubstrate compounds having the following formula:

wherein, R₁═H, R₆, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, NHC(O)R₆, NHC(O)OR₆,Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N˜OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein R₂═H, R₆, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, NHC(O)R₆, NHC(O)OR₆, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂,C(N˜OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein R₄═H, R₆, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, NHC(O)R₆, NHC(O)OR₆,Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N˜OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein R₅═H, R₆, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, NHC(O)R₆, NHC(O)OR₆, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂,C(N˜OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₃═NO₂, CHO, (CR₈═CR₈)_(k)CN or (CR₈═CR₈)_(k) NO₂, where k isan from 1 to 3, or

wherein, R₆═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₇═R₆, OR₆, orN(R₆)₂; wherein R₈═H or (CH₂)_(n)CH₃; where n is an integer from 0 to 3.

Also provided are chromogenic sialidase substrate compounds having theformula of General Structure I, wherein, R₁═H, R₆, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N˜OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein, R₂ or R₄═H, R₆, OR₆,OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂,C(N˜OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₃═H, R₆, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO,CO₂R₆, C(O)N(R₆)₂, C(N˜OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆,OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0to 3; wherein, R₅═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO,CO₂R₆, C(O)N(R₆)₂, C(N˜OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆,OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0to 3; wherein, R₂ or R₄═NO₂, CHO, (CR₈═CR₈)_(k)CN or (CR₈═CR₈)_(k)NO₂,where k is an integer from 1 to 3, or

wherein, R₆═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₇═R₆, OR₆, orN(R₆)₂; wherein R₈═H or (CH₂)_(n)CH₃; where n is an integer from 0 to 3.

Also provided are chromogenic sialidase substrate compounds having theformula of General Structure I, wherein, R₁ or R₅═H, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N˜OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein, R₂═H, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N˜OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein, R₃═H, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N˜OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein, R₄═H, OR₆, OC(O)R₇, NO₂,NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆, C(O)N(R₆)₂, C(N˜OH)NH₂, OPO₃R₆,OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆, OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN,where j is an integer from 0 to 3; wherein R₁ or R₅═NO₂, CHO,(CR₈═CR₈)_(k)CN or (CR₈═CR₈)_(k)NO₂, where k is an integer from 1 to 3;wherein, R₆═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₇═R₆, OR₆, orN(R₆)₂, wherein R₈═H or (CH₂)_(n)CH₃; where n is an integer from 0 to 3.

Also provided are chromogenic sialidase substrate compounds having thefollowing formula:

wherein, R₁═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆,C(O)N(R₆)₂, C(N˜OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₂═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆,C(O)N(R₆)₂, C(N˜OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₃═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆,C(O)N(R₆)₂, C(N˜OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₄═H, OR₆, OC(O)R₇, NO₂, NH₂, N(R₆)₂, Cl, Br, I, F, CHO, CO₂R₆,C(O)N(R₆)₂, C(N˜OH)NH₂, OPO₃R₆, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₆,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₆, or CN, where j is an integer from 0 to 3;wherein, R₅═H or (CH₂)_(k)CH₃, where k is an integer from 0 to 4;wherein, R₆═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₇═R₆, OR₆, orN(R₆)₂.

Also provided are chromogenic sialidase substrate compounds having thefollowing formula:

wherein, R₁═H, OR₃, OC(O)R₄, NO₂, NH₂, N(R₃)₂, Cl, Br, I, F, CHO, CO₂R₃,C(O)N(R₃)₂, C(N˜OH)NH₂, OPO₃R₃, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₃, OSO₃R₃,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₃, or CN, where j is an integer from 0 to 3;wherein, R₂═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein R₃═H, C(CH₃)₃,CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or (CH₂)_(m)CH₃, where m isan integer from 0 to 3; wherein, R₄═R₃, OR₃, or N(R₃)₂.

Also provided are chromogenic sialidase substrate compounds having thefollowing formula:

wherein, R₁═H, OR₃, OC(O)R₄, NO₂, NH₂, N(R₃)₂, Cl, Br, I, F, CHO, CO₂R₃,C(O)N(R₃)₂, C(N˜OH)NH₂, OPO₃R₃, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₃, OSO₃R₃,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₃, or CN, where j is an integer from 0 to 3;wherein, R₂═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein R₃═H, C(CH₃)₃,CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or (CH₂)_(m)CH₃, where m isan integer from 0 to 3; wherein, R₄═R₃, OR₃, or N(R₃)₂.

Also provided are chromogenic sialidase substrate compounds having thefollowing formula:

wherein, R₁═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N˜OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₂═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N˜OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₃═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N˜OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₄═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N˜OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₅═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N˜OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₆═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N˜OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₇═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N˜OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₆, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein R₈═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₉═R₈, OR₈, orN(R₈)₂.

Also provided are chromogenic sialidase substrate compounds having thefollowing formula:

wherein, R₁═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N˜OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₂═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N˜OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₃═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N˜OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₄═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N˜OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₅═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N˜OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₆═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N˜OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where is an integer from 0 to 3;wherein, R₇═H, OR₈, OC(O)R₉, NO₂, NH₂, N(R₈)₂, Cl, Br, I, F, CHO, CO₂R₈,C(O)N(R₈)₂, C(N˜OH)NH₂, OPO₃R₈, OPO₂(CH₂)_(j)CH₃, CH₂PO₃R₈, OSO₃R₈,OSO₂(CH₂)_(j)CH₃, CH₂SO₃R₈, or CN, where j is an integer from 0 to 3;wherein, R₈═H, C(CH₃)₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)(CH₂)_(m)CH₃, or(CH₂)_(m)CH₃, where m is an integer from 0 to 3; wherein, R₉═R₈, OR₈, orN(R₈)₂.

The subject invention further pertains to analogs, salts, derivatives,and mixtures of the subject compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a. A red color change produced by the substrate compound 14 (a) nosialidase added (left), (b) with sialidase added (right).

FIG. 1b. An orange color change produced by the substrate compound 11(a) no sialidase added (left), (b) with sialidase added (right).

FIG. 2—synthetic approaches for selected examples from General StructureI are summarized in this reaction scheme.

FIG. 3—synthetic approaches for selected examples from General StructureII are summarized in this reaction scheme.

FIG. 4—synthetic approaches for selected examples from GeneralStructures IIIa and IIIb are summarized in this reaction scheme.

FIG. 5—synthetic approaches for selected examples from GeneralStructures IVa and IVb are summarized in this reaction scheme.

FIG. 6—shows an overall scheme for the preparation of methylN-acetyl-β-D-neuraminate (2), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3),methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formylphenyl)-α-D-neuraminate(4), and N-acetyl-2-O-(4-formylphenyl)-β-D-neuraminic acid (5).

FIG. 7—shows an overall scheme for the preparation ofN-acetyl-2-O-[4-(2-nitrovinyl)phenyl]-α-D-neuraminic acid (6).

FIG. 8—shows an overall scheme for the preparation of4-hydroxy-2-methoxybenzaldehyde (8), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminate(9), and N-acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminic acid(10).

FIG. 9—shows an overall scheme for the preparation ofN-acetyl-2-O-[3-methoxy-4-(2-nitrovinyl)phenyl]-α-D-neuraminic acid(11).

FIG. 10—shows an overall scheme for the preparation of methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicacid (12) and N-acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicacid (13).

FIG. 11—shows an overall scheme for the preparation ofN-acetyl-2-O-[2-methoxy-4-(2-nitrovinyl)phenyl]-α-D-neuraminic acid(14).

FIG. 12—shows the overall scheme for preparation ofN-acetyl-2-O-(5-bromo-4-chloroindol-3-yl)-α-D-neuraminic acid (28).

This application contains at least one drawing executed in color. Copiesof the patent issuing from this application with color drawings will beprovided by the U.S. Patent Office upon request and payment of thenecessary fee.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention pertains to materials and methods useful fordetecting sialidase. Sialidase is an enzyme known to be associated witha variety of pathological conditions. Sialidases are produced bybacteria, viruses, and protozoa; therefore, detecting the presence ofsialidase in a biological sample can be indicative of the presence ofthese microbes. In specific embodiments, the detection of sialidases canbe performed according to the subject invention in order to identifyvaginal and periodontal infections, as well as to detect Pseudomonasaeruginosa in cystic fibrosis patients.

The presence of sialidase is detected according to the subject inventionthrough the use of novel chromogenic substrate compounds. Thesecompounds advantageously provide a visible color change when acted uponby sialidase. Thus, these substrates, when utilized according to theteachings of the subject invention, can be used to easily and accuratelydetect the presence of sialidase in a sample. In a preferred embodiment,the sample which is tested is a biological sample such as blood, mucous,saliva, and the like.

The subject invention provides compounds having structures as shown inGeneral Structures I, II, IIIa, IIIb, IVa, and IVb. The inventionfurther includes derivatives, analogs, and salts of the exemplifiedcompounds. These derivatives, analogs, and salts, which can readily beprepared by one skilled in the art and having the benefit of the instantdisclosure, fall within the scope of the present invention so long assuch compounds have the characteristic of producing a color change whenacted upon by a sialidase enzyme.

The compounds of the subject invention can be employed in a wide varietyof assay formats. Typically, the assay will involve contacting a sampleto be tested for the presence of sialidase with a chromogenic enzymesubstrate of the subject invention. A color change occurring after thesample is contacted with the substrate is indicative of the presence ofsialidase. The assay may optionally utilize positive and/or negativecontrols to aid in the interpretation and verification of the results.The results may also be quantified using standard optical measuringinstrumentation.

Materials and Methods

Biochemical Evaluation for the Chromogenic Product of SialidaseSubstrate Compounds. Sialidase can be obtained from, for example,purified recombinant bacterial sialidase from Salmonella T., wholeinfluenza virus, or culture medium containing secreted human sialidasefrom 2CFSME0 cell line. The sialidase preparation is added to a bufferof 0.1 M sodium acetate at pH6.0, and the substrate compound is providedat about 0.5 mM concentration. The reaction takes place in roomtemperature for 20 mins in a volume of 100 μl. At the end of thereaction, the pH is adjusted by adding a solution (0.2 M glycine, andsodium hydroxide with a pH value of 11.0). A color change is readilyvisible as exemplified by FIGS 1 a and 1 b. The color change can bequantitated by measuring the light absorption of the reaction mixture.

FIG. 1a shows a red color change produced by the substrate compound 14(a) no sialidase added (left), (b) with sialidase added (right). FIG. 1bshows an orange color change produced by the substrate compound 11 (a)no sialidase added (left), (b) with sialidase added (right).

General Methodologies. The following general methods are applicable tothe synthesis of compounds of the invention. Modifications or variationsof these methods can readily be utilized by those skilled in the arthaving the benefit of the instant disclosure.

Esterification. N-Acetyl-D-neuraminic acid is treated withmethanol-washed Dowex 50W-X4 in methanol with stirring at roomtemperature for a period of time, generally 4 h. The mixture isfiltered, and the filtrate is concentrated to give the desiredesterified product after crystallization.

Those skilled in the art would recognize that other standard proceduresare available for esterification of the same material, such as the useof other cation exchange resins, e.g., Amberlyst 15 or Dowex 50W-X8,among others.

O-Acetylation and Glycosyl Chloride Preparation. Treatment of theesterified product with acetyl chloride with stirring at roomtemperature under anhydrous conditions for a period of time, generally20-24 h, results in formation of the per-O-acetylated glycosyl chloride.Note that in some instances the bubbling of dry hydrogen chloride (gas)into the reaction vessel is necessary to effect glycosyl chlorideformation. Concentration of the reaction mixture with the water bathtemperature not exceeding 35° C., and drying the residue in vacuoprovides the product as a foam sufficiently pure for subsequentreactions.

Those skilled in the art would recognize that other standard proceduresare available for O-acetylation and glycosyl chloride preparation of thesame material, including a previously reported two-step procedure (Kuhn,et al., 1966) which involves per-O-acetylation of the same material withacetic anhydride in perchloric acid, followed by formation of theglycosyl chloride by treatment with acetyl chloride.

O-Glycosylation. Treatment of the substituted hydroxybenzaldehydederivative with sodium hydride in tetrahydrofuran with stirring at roomtemperature for a period of time, generally 1-3 h, results in formationof the sodium salt. Subsequent treatment of the sodium salt with theglycosyl chloride (compound 3) with stirring, for a period of time,generally 12-60 h, at room temperature results in O-glycosylation.Concentration of the reaction mixture, treatment of the residue withethyl acetate and water, separation and drying of the organic phase,concentration of the organic phase, and column chromatography of thecrude material affords the desired O-glycoside.

Those skilled in the art would recognize that other standard proceduresare available for O-glycosylation of the same materials, such astraditional Lewis Acid-mediated O-glycosylation methodologies (Okamotoand Goto, 1990), as well as the use of alternate salts of thesubstituted aromatic hydroxyl derivative, including tetrabutylammonium(Baggett and Marsden, 1982) or silver (Holmquist and Brossmer, 1972)salts, among others.

De-O-acetylation and De-esterification. The protected O-glycoside istaken up in aqueous sodium hydroxide and stirred at room temperature fora period of time, generally 1-4 h. The mixture is then adjusted to pH3-5 with Dowex 50W-X4 (H+) resin. Filtration, followed by lyophilizationof the filtrate affords the desired de-O-acetylated and de-esterifiedmaterial.

Those skilled in the art would recognize that other standard proceduresare available for the complete de-O-acetylation and de-esterification ofthe same material, including a two-step procedure which involvescomplete de-O-acetylation of the same material with sodium methoxide inmethanol or with an appropriate ion exchange resin, e.g. AmberliteIRA-400 (OH−), followed by de-esterification using conditions of acidhydrolysis or base hydrolysis.

Synthesis of Chromogenic Substrates of Sialidases

A. Compounds with General Structure I and their salts and derivatives,may be prepared using any of several methods known in the art for thesynthesis of substituted sialic acid analogs containing analogousstructures.

To illustrate, synthetic approaches for selected examples (FIG. 2) fromGeneral Structure I are summarized in the following reaction scheme andare representative of the types of procedures which can be employed.Table 1 lists specific compounds, the synthesis of which is exemplifiedherein.

TABLE 1

Compound R₁ R₂ R₃ 5 H H CHO 6 H H CH═CHNO₂ 10 H OCH₃ CHO 11 H OCH₃CH═CHNO₂ 13 OCH₃ H CHO 14 OCH₃ H CH═CHNO₂

In another specific embodiment, the subject invention includes compoundshaving the following structures:

Advantageously, these compounds produce a blue color change when actedupon by a sialidase.

FIG. 2 illustrates constructing a basic skeleton of General Structure Ivia acid-mediated esterification of commercially availableN-acetyl-D-neuraminic acid (1) to provide methyl N-acetyl-D-neuraminate(2), and subsequent per-O-acetylation and generation of the glycosylchloride (3) according to modifications of known procedures (Kuhn etal., 1966; Ogura et al., 1986; Patel and Richardson, 1986). Treatment ofcompound 3 with the sodium salt of numerous substitutedhydroxybenzaldehyde derivatives would provide the key intermediates tothe desired targets (compounds 4, 9, and 12). Generation of the sodiumsalt would be accomplished with sodium hydride in tetrahydrofuran. Thismethod of O-glycosylation has already been applied in thestereoselective preparation of numerous -O-glycosides ofN-acetyl-D-neuraminic acid (Myers, et al., 1980; Eschenfelder andBrossmer, Carbohydr. Res., 1987; Eschenfelder and Brossmer,Glycoconjugate J., 1987; Okamoto and Goto, 1990; Warner and O'Brien,1979) derived from aromatic hydroxyls. However, none of the productsdescribed herein are contained in the aforementioned references.Synthetic approaches to or references to synthetic approaches tointermediates (1) and (2) are contained in the aforementionedreferences. Subsequent de-O-acetylation and de-esterification of theresulting intermediates can be accomplished with an aqueous sodiumhydroxide solution and workup involving acidification of the reactionmedium. This provides access to the formyl substitutedphenolic-O-glycosides (compounds 5, 10, and 13).

Treatment of the derived targets (compounds 5, 10, and 13) withnitromethane, ammonium acetate, and acetic acid in ethanol under refluxprovides access to the desired nitrovinyl targets (compounds 6, 11, and14). This procedure has been utilized in the preparation of nitrovinylanalogs of other monosaccharides (Patel and Richardson, 1986; Aamlid, etal., 1990) as chromogenic substrates for the assay of glycosidases;however, none of the products or intermediates described herein arecontained in the aforementioned references.

It should also be noted that the p-nitrophenyl O-glycoside ofN-acetylneuraminic acid (General Structure I, wherein, R₁═R₂═R₄═R₅═H andR₃═NO₂ has been reported as a chromogenic substrate of sialidases(Eschenfelder and Brossmer, Carbohydr. Res., 1987). Condensation ofcompounds 5, 10, or 13 with any of numerous aromatic keto compounds inthe presence of ammonia and ammonium chloride, provides ready access tonumerous chromogenic substrates of sialidases (for representativeexamples, see compounds 15 and 16 FIG. 2) of General Structure I.

B. Compounds with General Structure II and their salts and derivatives,may be prepared using any of several methods known in the art for thesynthesis of substituted sialic acid analogs containing analogousstructures.

To illustrate, synthetic approaches for selected examples from GeneralStructure II (FIG. 3) are summarized in the following reaction schemeand are representative of the types of procedures to be employed. FIG. 3illustrates constructing a basic skeleton of General Structure II viaacid-mediated esterification of commercially availableN-acetyl-D-neuraminic acid (1) to provide methyl N-acetyl-D-neuraminate(2), and subsequent per-O-acetylation and generation of the glycosylchloride (3) according to modifications of known procedures (Kuhn etal., 1966; Ogura et al., 1986; Patel and Richardson, 1986). Treatment ofany of numerous substituted indoxyl 1,3-diacetate compounds (compound17) with sodium methoxide in anhydrous N,N-dimethylformamide readilyprovides the modified 3-hydroxy indole (compound 18). This procedure hasbeen utilized in the preparation of 5-bromo-3-hydroxyindole (compound18, wherein, R₁═R₃═R₄═H and R₂═Br) (Eschenfelder and Brossmer,Glycoconjugate J., 1987). Subsequent treatment of the compound 18 withcompound 3 in anhydrous N,N-dimethylformamide provides the desiredmodified indole O-glycoside (compound 19) according to a known procedurefor the preparation of methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(5-bromoindol-3-yl)-α-D-neuraminate(compound 19, wherein, R₁═R₃═R₄═R₅═H and R₂═Br) (Eschenfelder andBrossmer, Glycoconjugate J., 1987). Analogously, 3-indolyl O-glycosidesof other monosaccharides have been prepared using these and alternateconditions (Robertson, 1927; Freudenberg, et al., 1952; Anderson andLeeback, 1961; Horwitz, et al., 1964; Ley, et al., 1987); however, noneof the products or intermediates described herein are contained in theaforementioned references. Treatment of compound 19 with sodium hydridein tetrahydrofuran, followed with an alkyl halide (R₅Br) would providethe N-alkylated product. Subsequent de-O-acetylation andde-esterification of the resulting intermediates can be accomplishedwith an aqueous sodium hydroxide solution and workup involvingacidification of the reaction medium. This provides access to thesubstituted indole -O-glycosides (compound 20). It should be noted thatN-acetyl-2-O-(5-bromoindol-3-yl)-α-D-neuraminic acid (compound 20,wherein, R₁═R₃═R₄═R₅═H and R₂═Br) has been utilized as a chromogenicsubstrate for sialidases of many different origins (Eschenfelder andBrossmer, Glycoconjugate J., 1987).

C. Compounds with General Structures IIIa and IIIb and their salts andderivatives, may be prepared using any of several methods known in theart for the synthesis of substituted sialic acid analogs containinganalogous structures.

To illustrate, synthetic approaches for selected examples from GeneralStructures IIIa and IIIb are summarized in FIG. 4 and are representativeof the types of procedures to be employed. FIG. 4 illustratesconstructing a basic skeleton of General Structures IIIa/IIIb viaacid-mediated esterification of commercially availableN-acetyl-D-neuraminic acid (1) to provide methyl N-acetyl-D-neuraminate(2), and subsequent per-O-acetylation and generation of the glycosylchloride (3) according to modifications of known procedures (Kuhn etal., 1966; Ogura et al., 1986; Patel and Richardson, 1986). Treatment ofcompound (3) with the sodium salt of numerous substituted coumarinderivatives provides the key intermediates to the desired targets(compound 21). Generation of the sodium salt can be accomplished withsodium hydride in tetrahydrofuran. This method of O-glycosylation hasalready been applied in the stereoselective preparation of numerous-O-glycosides on N-acetyl-D-neuraminic acid (Myers, et al., 1980;Eschenfelder and Brossmer, Carbohydr. Res., 1987; Eschenfelder andBrossmer, Glycoconjugate ., 1987; Okamoto and Goto, 1990; Warner andO'Brien, 1979) derived from aromatic hydroxyls, including specificexamples for the preparation of a substituted coumarin O-glycoside(compound 21, wherein, R₁═H and R₂═CH₃) (Warner and O'Brien, 1979;Myers, et al., 1980). Subsequent de-O-acetylation and de-esterificationof the resulting intermediates can be accomplished with an aqueoussodium hydroxide solution and workup involving acidification of thereaction medium. This provides access to the modified coumarinO-glycosides (compound 22).

D. Compounds with General Structures IVa and IVb and their salts andderivatives, may be prepared using any of several methods known in theart for the synthesis of substituted sialic acid analogs containinganalogous structures.

To illustrate, potential synthetic approaches for selected examples fromGeneral Structures IVa and IVb are summarized in FIG. 5 and arerepresentative of the types of procedures which can be employed. FIG. 5illustrates constructing a basic skeleton of General Structure IVa/IVbvia acid-mediated esterification of commercially availableN-acetyl-D-neuraminic acid (1) to provide methyl N-acetyl-D-neuraminate(2), and subsequent per-O-acetylation and generation of the glycosylchloride (3) according to modifications of known procedures (Kuhn etal., 1966; Ogura et al., 1986; Patel and Richardson, 1986). Treatment ofcompound (3) with the sodium salt of numerous substituted naphtholderivatives would provide the key intermediates to the desired targets(compound 23). Generation of the sodium salt can be accomplished withsodium hydride in tetrahydrofuran. This method of O-glycosylation hasalready been applied in the stereoselective preparation of numerous-O-glycosides on N-acetyl-D-neuraminic acid (Myers, et al., 1980;Eschenfelder and Brossmer, Carbohydr. Res., 1987; Eschenfelder andBrossmer, Glycoconjugate J., 1987; Okamoto and Goto, 1990; Warner andO'Brien, 1979) derived from aromatic hydroxyls. However, none of theproducts described herein are contained in the aforementionedreferences. Synthetic approaches to, or references to syntheticapproaches to, intermediates (1) and (2) are contained in theaformentioned references. Subsequent de-O-acetylation andde-esterification of the resulting intermediates can be accomplishedwith an aqueous sodium hydroxide solution and workup involvingacidification of the reaction medium. This would provide access to themodified naphthyl O-glycosides (compounds 24).

F. Biochemical Evaluation for the Chromogenic Product of the SialidaseSubstrate Compound. The source of sialidase was from purifiedrecombinant bacterial sialidase from Salmonella T., whole influenzavirus, or culture medium containing secreted human sialidase from2CFSME0 cell line. The sialidase preparation was added to a buffer of0.1 M sodium acetate at pH6.0, and the substrate compound 14 wasprovided at about 0.5 mM concentration. The reaction took place in roomtemperature for 20 mins in a volume of 100 μl. At the end of thereaction, the pH was adjusted by adding a solution (0.2 M glycine, andsodium hydroside with a pH value of 11.0). A color change to red wasreadily visible as examplified by FIGS. 1a and 1 b. The color change wasquantitated by measuring the light absorption of the reaction mixture.The light absorption was scanned with a photospectrometer. The peakvalue for compound IBX4010 is 495 nm. At a substrate concentration of0.2 mM, the light absorption at 495 nm with a 1 cm path is 1.203. Thecontrol in which the reaction mixture was kept under the same conditionfor 10 minutes without addition of any enzyme had an absorption of 0.282at 495 nm with a 1 cm path.

Compound 11 was tested by the same method. At the end of the reactionwith pH adjusment, a color change to orange was readily visible asexemplified by FIGS. 1a and 1 b. The color change was quantitated bymeasuring the light absorption of the reaction mixture. Ten minutesafter the reaction, the mixture of the reaction product was adjusted tobasic pH and the light absorption was scanned with a photospectrometer.The peak value for compound IEBX4023 is 480 nm. At a substrateconcentration of 0.2 mM, the light absorption at 480 nm with a 1 cm pathis 4.065. The control in which the reaction mixture was kept under thesame condition for 10 minutes without addition of any enzyme had anabsorption of 1.452 at 480 nm with a 1 cm path.

G. Classes of Chromogenic Substrate Compounds of Sialidases. As usedherein, the “effective amount” of a compound of the invention requiredfor the use in the method presented herein will differ not only with theparticular compound to be selected but also with the mode ofapplication, and the nature of the sample specimen. The exact amountwill be evaluated by testing with a sufficient number of clinicalsamples in each application as conducted by persons skilled in the art.However, a generally suitable concentration will range from about 0.1 toabout 10 mM/ml of testing solutions. Furthermore, the compounds may beused as pure chemical applied to a test solution, or as a purechemically acceptable salt or derivative. However, it is preferable toprovide the active chemical or its chemically acceptable salt orderivative, as a medicinal formulation, either as a dry material(reaction solution provided seperately), or as a solution or suspension(an aqueous solution or other chemically acceptable solvent solutions),or as a dip stick. The subject specimen can be applied to the test formeasuring the activity levels of sialidases. Those skilled in the arthaving the benefit of the instant disclosure will appreciate thatamounts and modes of application are readily determinable without undueexperimentation.

The following detailed examples for methods of preparation are forillustration only, and are not intended to represent a limitation of theinvention. The structures of the compounds whose preparations aredescribed below are summarized in Table 1 for modified phenolderivatives and in FIG. 3 (for a single example where R₁═Cl; R₂═Br;R₃═R₄═R₅═H). In all cases synthetic intermediates and products werefound to be pure according to standards known to those skilled in theart (such as thin layer chromatography, melting or boiling points, gaschromatography, ion exchange chromatography, and/or high pressure liquidchromatography, elemental analysis, and spectroscopic methods).Furthermore, structures were characterized and assigned by spectroscopicmethods considered standard practices by those skilled in the art (suchas infrared, ultraviolet, and mass spectroscopies, ¹H and ¹³C nuclearmagnetic resonance spectroscopy, and/or x-ray crystallography). Selectedspectral data are described for intermediates and products.

EXAMPLE 1 Preparations of Methyl N-acetyl-β-D-neuraminate (2), MethylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3),MethylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formylphenyl)-α-D-neuraminate(4), and N-acetyl-2-O-(4-formylphenyl)-α-D-neuraminic Acid (5)

The overall scheme is shown in FIG. 6.

Preparation of Methyl N-acetyl-β-D-neuraminate (2). To a stirredsuspension of N-acetylneuraminic acid (1) (10.0 g, 32.3 mmol) inmethanol (1.0 L) was added methanol-washed Dowex 50W-X4 (25.0 g) under anitrogen atmosphere at room temperature protected from light. Theresulting mixture was allowed to stir at room temperature for 4 h. Themixture was filtered and the filtrate was concentrated to dryness. Theresidue was crystallized from methanol to afford pure compound (2) (10.1g, 96%): mp 178-180° C. (d). A literature reference (Kuhn et al., 1966)reports mp 179-180° C. A second literature reference (Ogura et al.,1986) reports mp 180-182° C.

¹H NMR (D₂O): (1.73 (dd, 1H, J_(3a,4) 12.0 Hz, J_(3a,3e) 13.3 Hz, H-3a),1.87 (s, 3H, NAc), 2.14 (dd, 1H, J_(3e,4) 5.0 Hz, H-3e), 3.33-3.48 (m,2H), 3.52-3.58 (m, 1H), 3.62-3.68 (m, 1H), 3.65 (s, 3H, CO₂CH₃),3.69-3.76 (m, 1H), 3.84-3.92 (m, 2H).

Preparation of MethylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3). Asuspension of compound (2) (3.67 g, 11.3 mmol) in acetyl chloride (225mL) was stirred under anhydrous conditions at room temperature protectedfrom light for 24 h. The resulting solution was concentrated to dryness,the residue was coevaporated with anhydrous ether (2×50 mL), followed bycoevaporations with anhydrous benzene (2×50 mL). Note. that in allevaporations, the water bath temperature was maintained at or below 35°C. The residue was dried in vacuo to afford pure compound (3) (4.8 g,83%) as a syrup. A literature reference (Ogura et al., 1986) reports mp116-118° C.; whereas, a second literature reference (Kuhn et al., 1966)reports compound (3) as a syrup.

¹H NMR (CDCl₃): (1.92 (s, 3H, NAc), 2.06, 2.07, 2.10, 2.14 (4 s, 12H,4×OAc), 2.28 (dd, 1H, J_(3a,4) 11.3 Hz, J_(3a,3e) 13.5 Hz, H-3a), 2.79(dd, 1H, J_(3e,4) 4.5 Hz, H-3e), 3.89 (s, 3H, CO₂CH₃), 4.07 (dd, 1H,J_(8,9′) 5.6 Hz, J_(9′,9″) 11.6 Hz, H-9′), 4.13-4.28 (m, 1H, H-5), 4.36(dd, 1H, J_(6,7) 2.5 Hz, J_(5,6) 10.5 Hz, H-6), 4.43 (dd, 1H, J_(8,9″)2.9 Hz, H-9″), 5.18 (ddd, 1H, J_(7,8) 6.7 Hz, H-8), 5.40 (ddd, 1H,J_(4,5) 10.4 Hz, H-4), 5.49-5.52 (m, 2H, H-7, NH).

Preparation of MethylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formylphenyl)-α-D-neuraminate(4). To a stirred solution of 4-hydroxybenzaldehyde (121 mg, 0.98 mmol)in anhydrous tetrahydrofuran (6.0 mL) was added portionwise sodiumhydride (48 mg of a 60% dispersion in mineral oil, 1.2 mmol) under anitrogen atmosphere at room temperature. The resulting mixture wasallowed to stir at room temperature for 25 min. The mixture was treatedwith compound (3) (500 mg, 0.98 mmol) and the resulting mixture wasstirred under a nitrogen atmosphere at room temperature for 52 h. Themixture was concentrated to dryness, the residue was diluted with ethylacetate (15 mL), and washed with water (15 mL). The aqueous phase wasextracted with ethyl acetate (3×15 mL), and combined organic phases weredried with magnesium sulfate, filtered, and the filtrate wasconcentrated to dryness. The residue was chromatographed (silica gel,1:1 acetone-hexanes as eluting solvent) to afford pure compound (4) (238mg, 41%): R_(f)═0.26 (1:1 acetone-hexanes; UV, H₂SO₄).

¹H NMR (CDCl₃): (1.95 (s, 3H, NAc), 2.07, 2.09, 2.13, 2.22 (4 s, 12H,4×OAc), 2.32 (ut, 1H, J_(3a,3e)=J_(3a,4)=13.2 Hz, H-3a), 2.77 (dd, 1H,J_(3e,4) 5.0 Hz, H-3a), 3.67 (s, 3H, CO₂CH₃), 4.10-4.22 (m, 2H),4.24-4.32 (m, 1H), 4.63 (dd, 1H, J 2.0 Hz, J 11.7 Hz), 4.97-5.06 (m,1H), 5.30 (d, 1H, J 12.6 Hz), 5.41 (s, 2H), 7.20 (d, 2H, J 9.6 Hz,2×ArH), 7.86 (d, 2H, J 9.6 Hz, 2×ArH), 9.95 (s, 1H, CHO).

Preparation of N-Acetyl-2-O-(4-formylphenyl)-α-D-neuraminic acid (5). Asolution of compound (4) (171 mg, 0.29 mmol) in aqueous sodium hydroxide(5.0 mL of a 1.0 M solution, 5.0 mmol) was stirred at room temperaturefor 2 h. The resulting mixture was cooled to 0° C. and treated withmethanol-washed Dowex 50W-X4 til pH 3. The mixture was filtered, thefiltered resin was rinsed with water, and the filtrate was lyophilizedto afford compound (5) (118 mg, 99%): R_(f)=0.31 (5:2:1 ethylacetate-methanol-0.02% aqueous calcium chloride; UV, H₂SO₄).

¹H NMR (D₂O): (2.08 (s, 3H, NAc), 2.05-2.12 (m, 1H, H-3a), 2.84 (dd, 1H,J_(3e,4) 5.6 Hz, J_(3a,3e) 13.1 Hz, H-3e), 3.58-3.69 (m, 2H), 3.82-3.90(m, 3H), 3.92-4.10 (m, 1H), 4.21 (dd, 1H, J 1.9 Hz, J 11.3 Hz), 7.32 (d,2H, J 9.6 Hz, 2×ArH), 7.92 (d, 2H, J 9.6 Hz, 2×ArH), 9.84 (s, 1H, CHO).

EXAMPLE 2 Preparation ofN-acetyl-2-O-[4-(2-nitrovinyl)phenyl]-α-D-neuraminic Acid

The overall reaction scheme is shown in FIG. 7. For the preparations ofmethyl N-acetyl-β-D-neuraminate (2), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3),methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formylphenyl)-α-D-neuraminate(4), and N-acetyl-2-O-(4-formylphenyl)-α-D-neuraminic acid (5), see theexperimental details for Example 1.

To a stirred solution of compound (5) (50 mg, 0.10 mmol) in a mixture ofethanol (2.0 mL) and acetic acid (0.05 mL) was added ammonium acetate(50 mg, 0.65 mmol) and nitromethane (0.20 mL, 3.70 mmol) at roomtemperature. The reaction mixture was heated under reflux for 30 min,cooled to room temperature, and evaporated to dryness. The residue waschromatographed (silica gel, 5:2:1 ethyl acetate-methanol-0.02% aqueouscalcium chloride as eluting solvent) to afford pure compound (6) (32 mg,68%): R_(f)=0.50 (5:2:1 ethyl acetate-methanol-0.02% aqueous calciumchloride; UV, H₂SO₄).

¹H NMR (D₂O): (2.05 (s, 3H, NAc), 1.95-2.02 (m, 1H, H-3a), 2.89 (dd, 1H,J_(3e,4) 5.2 Hz, J_(3a,3e) 12.9 Hz, H-3e), 3.57-3.69 (m, 2H), 3.85-4.00(m, 4H), 4.06 (dd, 1H, J 1.5 Hz, J 10.5 Hz), 7.22 (d, 2H, J 9.0 Hz,2×ArH), 7.64 (d, 2H, J 9.0 Hz, 2×ArH) 7.83 (d, 1H, J 13.5 Hz,H-vinylic), 8.13 (d, 1H, J 13.5 Hz, H-vinylic).

EXAMPLE 3 Preparation of 4-Hydroxy-2-methoxybenzaldehyde (8), MethylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminate(9) and N-acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminic acid (10)

The overall reaction scheme is shown in FIG. 8.

For the preparation of methyl N-acetyl-α-D-neuraminate (2), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3), seethe experimental details presented previously.

Preparation of 4-Hydroxy-2-methoxybenzaldehyde (8). To a stirredsolution of 3-methoxyphenol (14.9 g, 120 mmol) in 15% aqueous potassiumhydroxide (500 mL) was added chloroform (100 mL). The resulting solutionwas heated under reflux for 4 h, cooled to room temperature, and treatedwith 10% aqueous hydrochloric acid til pH 4. The suspension wasfiltered, and the filter cake was rinsed with chloroform (150 mL). Thechloroform phase was separated, the aqueous phase was extracted withadditional portions of chloroform (3×50 mL), and the combined organicphases were dried with magnesium sulfate. The solution was then filteredthrough a short column (silica gel, chloroform as eluting solvent) toafford compound (8). Crystallization from ethyl acetate gave purecompound (8) (1.8 g, 10%): mp 150-152° C. A literature reference (Pateland Richardson, 1986) reports mp 154-156° C. Additional references(Tiemann and Koppe, 1881; de Kiewiet and Stephen, 1931) report mp 153°C.

¹H NMR (CDCl₃): (3.87 (s, 3H, OCH₃), 6.32-6.48 (m, 2H, 2×ArH), 7.62 (d,1H, J 9.6 Hz, ArH), 10.1 (s, 1H, CHO).

Preparation of MethylN-acetyl-4.7.8.9-tetra-O-acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminate(9). To a stirred solution of compound (8) (900 mg, 5.92 mmol) inanhydrous tetrahydrofuran (35 mL) was added portionwise sodium hydride(288 mg of a 60% dispersion in mineral oil, 7.2 mmol) under a nitrogenatmosphere at room temperature. The resulting mixture was allowed tostir at room temperature for 2.5 h. The mixture was treated withcompound (3) (2.33 g, 4.58 mmol) and the resulting mixture was stirredunder a nitrogen atmosphere at room temperature for 115 h. The mixturewas concentrated to dryness, the residue was diluted with ethyl acetate(40 mL), and washed with water (40 mL). The aqueous phase was extractedwith ethyl acetate (3×40 mL), and combined organic phases were driedwith magnesium sulfate, filtered, and the filtrate was concentrated todryness. The residue was chromatographed (silica gel, 1:1acetone-hexanes as eluting solvent) to afford pure compound (9) (1.23 g,43%): R_(f)=0.31 (1:1 acetone-hexanes; UV, H₂SO₄).

¹H NMR (CDCl₃): (1.96 (s, 3H, NAc), 2.07, 2.09, 2.14, 2.18 (4 s, 12H,4×OAc), 2.23-2.45 (m, 1H, H-3a), 2.74 (dd, 1H, J_(3e,4) 5.9 Hz,J_(3a,3e) 13.7 Hz, H-3e), 3.73 (s, 3H, CO₂CH₃), 3.93 (s, 3H, OCH₃),4.12-4.22 (m, 2H), 4.25-4.30 (m, 1H), 4.60 (dd, 1H, J 1.8 Hz, J 12.6Hz), 4.96-5.08 (m, 1H), 5.28-5.47 (m, 3H), 6.65 (d, 1H, J 3.0 Hz, ArH),6.74 (dd, 1H, J 3.0 Hz, J 9.6 Hz, ArH), 7.82 (d, 1H, J 9.6 Hz, ArH),10.33 (s, 1H, CHO).

Preparation of N-Acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminicacid (10). A solution of compound (9) (751 mg, 1.20 mmol) in aqueoussodium hydroxide (20.0 mL of a 1.0 M solution, 20.0 mmol) was stirred atroom temperature for 2 h. The resulting mixture was cooled to 0° C. andtreated with methanol-washed Dowex 50W-X4 til pH 3. The mixture wasfiltered, the filtered resin was rinsed with water, and the filtrate waslyophilized to afford compound (10) (288 mg, 54%): R_(f)=0.34 (5:2:1ethyl acetate-methanol-0.02% aqueous calcium chloride; UV, H₂SO₄).

¹H NMR (D₂O): (2.08 (s, 3H, NAc), 2.04-2.12 (m, 1H, H-3a), 2.87 (dd, 1H,J_(3,4) 5.6 Hz, J_(3a,3e) 15.0 Hz, H-3e), 3.60-3.68 (m, 2H), 3.81-3.95(m, 4H), 3.93 (s, 3H, OCH₃), 4.23 (dd, 1H, J 3.7 Hz, J 11.2 Hz), 6.85(dd, 1H, J 5.7 Hz, J 11.4 Hz, ArH), 6.97 (d, 1H, J 5.7 Hz, ArH), 7.75(d, 1H, J 11.4 Hz, ArH), 10.0 (s, 1H, CHO).

EXAMPLE 4 Preparation ofN-acetyl-2-O-[3-methoxy-4-(2-nitrovinyl)phenyl]-α-D-neuraminic Acid (11)

The overall reaction scheme is shown in FIG. 9. For the preparation ofmethyl N-acetyl-β-D-neuraminate (2), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3), seethe experimental details presented previously.

For the preparations of 4-hydroxy-2-methoxybenzaldehyde (8), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminate(9), and N-acetyl-2-O-(4-formyl-3-methoxyphenyl)-α-D-neuraminic acid(10), see the experimental details presented in Example 3.

To a stirred solution of compound (10) (120 mg, 0.27 mmol) in a mixtureof ethanol (4.8 mL) and acetic acid (0.12 mL) was added ammonium acetate(120 mg, 1.56 mmol) and nitromethane (0.48 mL, 8.86 mmol) at roomtemperature. The reaction mixture was heated under reflux for 30 min,cooled to room temperature, and evaporated to dryness. The residue waschromatographed (silica gel, 5:2:1 ethyl acetate-methanol-0.02% aqueouscalcium chloride as eluting solvent) to afford pure compound (11) (48mg, 36%): R_(f)=0.64 (5:2:1 ethyl acetate-methanol-0.02% aqueous calciumchloride; UV, H₂SO₄).

¹H NMR (D₂O): (2.06 (s, 3H, NAc), 1.98-2.03 (m, 1H, H-3a), 2.88 (dd, 1H,J_(3e,4) 5.1 Hz, J_(3a,3e) 14.0 Hz, H-3e), 3.59-3.68 (m, 2H), 3.75-4.00(m, 4H), 3.95 (s, 3H, OCH₃), 4.11 (dd, 1H, J 2.5 Hz, J 11.4 Hz), 6.82(d, 1H, J 10.8 Hz, ArH), 6.95 (br s, 1H, ArH), 7.54 (d, 1H, J 10.8 Hz,ArH), 8.00 (d, 1H, J 13.5 Hz, H-vinylic), 8.22 (d, 1H, J 13.5 Hz,H-vinylic).

EXAMPLE 5 Preparation of MethylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicAcid (12) and N-acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicAcid (13)

The overall reaction scheme is shown in FIG. 10. For the preparation ofmethyl N-acetyl-β-D-neuraminate (2), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3), seethe experimental details presented previously.

Preparation of MethylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicAcid (12). To a stirred solution of vanillin(4-hydroxy-3-methoxybenzaldehyde) (273 mg, 1.8 mmol) in anhydroustetrahydrofuran (12.0 mL) was added portionwise sodium hydride (86 mg ofa 60% dispersion in mineral oil, 2.2 mmol) under a nitrogen atmosphereat room temperature. The resulting mixture was allowed to stir at roomtemperature for 2.5 h. The mixture was treated with compound (3) (700mg, 1.38 mmol) and the resulting mixture was stirred under a nitrogenatmosphere at room temperature for 68 h. The mixture was concentrated todryness, the residue was diluted with ethyl acetate (25 mL), and washedwith water (25 mL). The aqueous phase was extracted with ethyl acetate(3×25 mL), and combined organic phases were dried with magnesiumsulfate, filtered, and the filtrate was concentrated to dryness. Theresidue was chromatographed (silica gel, chloroform, followed by ethylacetate as eluting solvent) to afford pure compound (12) (322 mg, 38%):R_(f)=0.64 (1:8 acetone-ethyl acetate; UV, H₂SO₄).

¹H NMR (CDCl₃): (1.94 (s, 3H, NAc), 2.08, 2.09, 2.14, 2.19 (4 s, 12H,4×OAc), 2.33 (ut, 1H, J_(3a,3e)=J_(3a,4)=13.5 Hz, H-3a), 2.82 (dd, 1H,J_(3e,4)5.4 Hz, H-3e), 3.70 (s, 3H, CO₂CH₃), 3.92 (s, 3H, OCH₃),4.10-4.18 (m, 2H), 4.23-4.31 (m, 1H), 4.52 (br d, 1H, J 11.4 Hz),4.97-5.12 (m, 1H), 5.20-5.28 (m, 1H), 5.30-5.40 (m, 2H), 7.32 (d, 1H,J9.0 Hz, ArH), 7.41-7.48 (m, 2H, 2×ArH), 9.92 (s, 1H, CHO).

Preparation of N-Acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicacid (13). A solution of compound (12) (236 mg, 0.38 mmol) in aqueoussodium hydroxide (6.0 mL of a 1.0 M solution, 6.0 mmol) was stirred atroom temperature for 160 min. The resulting mixture was cooled to 0° C.and treated with methanol-washed Dowex 50W-X4 til pH 3. The mixture wasfiltered, the. filtered resin was rinsed with water, and the filtratewas lyophilized to afford compound (13) (152 mg, 91%): R_(f)=0.46 (5:2:1ethyl acetate-methanol-0.02% aqueous calcium chloride; UV, H₂SO₄).

¹H NMR (D₂O): (2.08 (s, 3H, NAc), 2.03-2.12 (m, 1H, H-3a), 2.92 (dd, 1H,J_(3e,4) 5.2 Hz, J_(3a,3e) 13.9 Hz, H-3e), 3.54-3.70 (m, 2H), 3.80-4.20(m, 5H), 3.92 (s, 3H, OCH₃), 7.48 (d, 1H, J 9.6 Hz, ArH), 7.52-7.60 (m,2H, 2×ArH), 9.82 (s, 1H, CHO).

EXAMPLE 6 Preparation ofN-acetyl-2-O-[2-methoxy-4-(2-nitrovinyl)phenyl]-α-D-neuraminic Acid (14)

The overall reaction scheme is shown in FIG. 11. For the preparation ofmethyl N-acetyl-β-D-neuraminate (2), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3), seethe experimental details presented previously.

For the preparation of methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicacid (12) and N-acetyl-2-O-(4-formyl-2-methoxyphenyl)-α-D-neuraminicacid (13), see the experimental details presented in Example 5.

Preparation ofN-Acetyl-2-O-[2-methoxy-4-(2-nitrovinyl)phenyl]-α-D-neuraminic acid(14). To a stirred solution of compound (13) (25 mg, 0.06 mmol) in amixture of ethanol (2.0 mL) and acetic acid (0.02 mL) was added ammoniumacetate (24 mg, 0.32 mmol) and nitromethane (0.10 mL, 1.9 mmol) at roomtemperature. The reaction mixture was heated under reflux for 30 min,.cooled to room temperature, and evaporated to dryness. The residue waschromatographed (silica gel, 5:2:1 ethyl acetate-methanol-0.02% aqueouscalcium chloride as eluting solvent) to afford pure compound (14) (19mg, 70%): R_(f)32 0.64 (5:2:1 ethyl acetate-methanol-0.02% aqueouscalcium chloride; UV, H₂SO₄).

¹H NMR (D₂O): (2.07 (s, 3H, NAc), 1.97-2.04 (m, 1H, H-3a), 2.87 (dd, 1H,J_(3e,4) 5.1 Hz, J_(3a,3e) 14.0 Hz, H-3e), 3.54-3.69 (m, 2H), 3.81-4.05(m, 5H), 3.93 (s, 3H, OCH₃), 7.45 (d, 1H, J 9.6 Hz, ArH), 7.48-7.55 (m,2H, 2×ArH), 8.02 (d, 1H, J 13.3 Hz, H-vinylic), 8.20 (d, 1H, J 13.3 Hz,H-vinylic).

EXAMPLE 7 Preparation ofN-acetyl-2-O-(5-bromo-4-chloroindol-3-yl)-α-D-neuraminic Acid (28)

The overall reaction scheme is shown in FIG. 12. For the preparation ofmethyl N-acetyl-β-D-neuraminate (2), methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminate (3), seethe experimental details presented previously.

reparation of 5-Bromo-4-chloro-3-hydroxyindole (26). To a stirredsolution of 5-bromo-4-chloroindoxyl 1,3-diacetate (25) (1.0 g, 3.03mmol) in anhydrous N,N-dimethylformamide (3 mL) was added sodiummethoxide (270 mg, 5.00 mmol). The resulting dark-colored reactionmixture was degassed with nitrogen (g) for 30 min at room temperature.

Preparation of methylN-acetyl-4,7,8,9-tetra-O-acetyl-2-O-(5-bromo-4-chloroindol-3-yl)-α-D-neuriminate(27). The reaction mixture of compound (26) in N,N-dimethylformamide wastreated with stirring with compound (3) (238 mg, 0.468 mmol) at roomtemperature under a nitrogen atmosphere protected from light. After 16h, the reaction mixture was concentrated under vacuum, coevaporated withxylenes (3×25 mL) to remove traces of N,N-dimethylformamide, treatedwith ethyl acetate (40 mL), and filtered. The filtrate was concentratedto a residue that was chromatographed (silica gel, 1:8 acetone-ethylacetate as eluting solvent) to provide compound 27.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

References

Hirst, G. K. (1941) “The Agglutination of Red Blood Cells by AllontoicFluid of Chick Embryos Infected with Influenza Virus,” Science 94:22-23.

Crennell, S. J. , et al. (1993) “Crystal Structure of a BacterialSialidase (from Salmonella typhimurium LT2) Shows the Same Fold as anInfluenza Virus Neuraminidase,” Proceedings of the National Academy ofScience USA 90(November):9852-9856.

Crennell, S., et al. (1994) “Crystal structure of Vibrio choleraeneuraminidase reveals dual lectin-like domains in addition to thecatalytic domain,” Structure 2(6):535-44.

Liu, C., et al. (1995) “Influenza type A virus neuraminidase does notplay a role in viral entry, replication, assembly, or budding,” J.Virol. 69:1099-1106.

Briselden, A. M., et al. (1992) “Sialidases (Neuraminidases) inbacterial vaginosis and bacterial vaginosis-associated microflora,” J.Clin. Microbiol. 30:663-666.

Liljemark, W. F., et al. (1989) “Effect of neuraminidase on theadherence to salivary pellicle of Streptococcus sangius andStreptpcoccus mitis,” Caries Res. 23:141-145.

Cross, G. A. and G. B. Takle (1993) “The surface trans-sialidase familyof Trypanosoma cruzi,” Annu. Rev. Microbiol. 47:385-411.

Cacalano, G. et al. (1992) “Production of the Pseudomonas aeruginosaNeuraminidase is increased under hyperosmolar conditions and isregulated by genes involved in alginate expression,” J. Clin. Invest.89:1866-1874.

Bratosin, D., et al. (1995) “Flow cytofluorometric analysis of young andsenescent human erythrocytes probed with lectins—evidence that sialicacids control their life-span,” Glycoconj. J. 12:258-267.

Bonten, E., et al. (1996) “Characterization of human lysosomalneuraminidase defines the molecular basis of the metabolic storagedisorder sialidosis,” Genes & Devel. 10:3156-3169.

Gornati, R., et al. (1997) “Activities of glycolipidglycosyltransferases and sialidases during the early development ofXenopus laevis,” Mol. Cell Biochem. 166:117-124.

Lentz, M. R., R. G. Webster, G. M. Air (1987) “Site-Directed Mutation ofthe Active Site of Influenza Neuraminidase and Implications for theCatalytic Mechanism,” Biochemistry 26:5351-5358.

Aamlid, K. H. , G. Lee, B. V. Smith, A. C. Richardson, R. G. Price(1990) “New Colormetric Substrates for the Assay of Glycosidases,”Carbohydr. Res. 205:c5-c9.

Baggett, N., B. J. Marsden (1982) “Reinvestigation of the Synthesis of4-Methylcoumarin-7-yl5-Acetamido-3,5-dideoxy-(-D-glycero-D-galacto-2-nonulopyranosidonicAcid, A Fluorimetric Substrate for Neuriminidase,” Carbohydr. Res.110:11-18.

Eschenfelder, V., R. Brossmer (1987) “Synthesis of p-Nitrophenyl5-Acetamido-3,5-dideoxy-(-D-glycero-D-galacto-2-nonulopyranosidonicAcid, a Chromogenic Substrate for Sialidases,” Carbohydr. Res.162:294-297.

Eschenfelder, V., R. Brossmer, (1987) “5-Bromo-indol-3-yl5-Acetamido-3,5-dideoxy-(-D-glycero-D-galactononulopyranosidonic Acid, aNovel Chromogenic Substrate for the Staining of Sialidase Activity,”Glycoconjugate J. 4171-178.

Freudenberg, K., H. Resnik, H. Boesenberg, D. Rasenack (1952) “Das ander Verholzung Beteiligte Fermentsystem,” Chem. Ber. 85:641-647.

Holmquist, L., R. Brossmer (1972) “Specificity of neuraminidase,synthesis and properties of the 2-aminoethyl α- and the 2-pyridyl α- andβ-glycosides of N-acetyl-D-neuraminic acid,” Hoppe-Seyler's Z. Physiol.Chem. 353:1346-1350.

Horwitz, J. P., J. Chua, R. J. Curby, A. J. Tomson, M. A. Darooge, B. E.Fisher, J. Mauricio, I. Klundt (1964) “Substrates for CytochemicalDemonstration of Enzyme Activity. I. Some Substituted3-Indolyl-(-D-glycopyranosides,” J. Med. Chem. 7:574-575.

de Kiewiet, T. E., H. Stephen (1931) “2-Hydroxy-4-methoxy- and4-Hydroxy-2-methoxy-benzaldehydes,” J. Chem. Soc. 133:84-85.

Kuhn, R., P. Lutz, D. L. MacDonald (1966) “Synthese anomererSialinsäure-methylketoside,” Chem. Ber. 99:611 -617.

Ley, A. N., R. J. Bowers, S. Wolfe (1988) “Indoxyl-(-D-glucuronide, aNovel Chromogenic Reagent for the Specific Detection and Enumeration ofEscherichia coli in Environmental Samples,” Can. J. Microbiol.34:690-693.

Myers, R. W., R. T. Lee, Y. C. Lee, G. H. Thomas, L. W. Reynolds, Y.Uchida (1980) “The Synthesis of 4-Methylumbelliferyl α-Ketoside ofN-Acetyl-Neuraminic Acid and Its Use in a Fluoromatic Assay forNeuraminidases,” Anal. Biochem. 101:166-174.

Ogura, H., K. Furuhata, M. Itoh, Y. Shitori (1986) “Syntheses of2-O-Glycosyl Derivatives of N-Acetyl-D-neuraminic Acid,” Carbohydr. Res.158:37-51.

Okamoto, K., T. Goto (1990) “Glycosidation of Sialic Acid,” Tetrahedron,46:5835-5857.

Patel, A., A. C. Richardson (1986) “3-Methoxy-4-(2-nitrovinyl)phenylGlycosides as Potential Chromogenic Substrates for the Assay ofGlycosidases,” Carbohydr. Res. 146:241-249.

Paulsen, H., P. Matschulat, “Synthese von C-Glycosiden derN-Acetylneuraminsäure und weiteren Derivaten,” Liebigs Ann. Chem.487-495.

Robertson, A. (1927) “Syntheses of Glucosides. Part I. The Synthesis ofIndican,” J Chem. Soc. 1937-1943.

Tiemann, F., P. Koppe (1981) “Ueber die Darstellung vonProtocatechualdehyd aus Brenzcatechin, sowie einige Derivate desGuajacols und Kreosols,” Chem Ber. 14:2015-2028.

Warner, T. G., J. S. O'Brien (1979) “Synthesis of2′-(4-Methylumbelliferyl)-(-D-N-acetylneuriminic Acid and Detection: ofSkin Fibroblast Neuriminidase in Normal Humans and in Sialidosis ,”Biochemistry 18:2783-2787.

What is claimed is:
 1. A method of detecting the presence of a sialidasein a sample comprising contacting a chromogenic sialidase substratecompound comprising the formula of General Structure I or a compositioncomprising a chromogenic sialidase substrate of General Structure I anddetecting a visible color change that indicates the presence of asialidase, wherein the chromogenic sialidase substrate compoundcomprises:

wherein R₁, R₂, R₄, or R₅ are substituents selected from the groupconsisting of H, R₆, Cl, Br, I, F, and NO₂, provided that at least twoof said substituents are substituted with H; R₃ is CH═CHNO₂,

R₆ is H, CH(CH₃)₂, (CH₂)_(m)CH₃ and m is an integer from 0 to 3; orsalts of said chromogenic sialidase substrate compounds.
 2. The methodaccording to claim 1, wherein R₁ and R₂, are H.
 3. The method accordingto claim 1, wherein R₁ and R₄, are H.
 4. The method according to claim1, wherein R₁ and R₅, are H.
 5. The method according to claim 1, whereinR₂ and R₄ are H.
 6. The method according to claim 1, wherein R₂ and R₅,are H.
 7. The method according to claim 1, wherein R₄ and R₅, are H. 8.The method according to claim 1, wherein R₁, R₂, and R₅ are H.
 9. Themethod according to claim 1, wherein R₁, R₄, and R₅ are H.
 10. Themethod according to claim 1, wherein R₂, R₄, and R₅ are H.
 11. Themethod according to claim 1, wherein R₁, R₂, R₄, and R₅ are H.
 12. Themethod according to claim 1, wherein R3 is CH═CHNO₂.
 13. The methodaccording to claim 1, wherein R₃ is


14. The method according to claim 1, wherein R₃ is


15. The method according to claim 1, wherein R₆ is H.
 16. The methodaccording to claim 1, wherein R₆ is CH(CH₃)₂.
 17. The method accordingto claim 1, wherein R₆ is (CH₂)_(m)CH₃ wherein m is an integer from 0 to3.
 18. The method according to claim 17, wherein m is
 0. 19. The methodaccording to claim 17, wherein m is
 1. 20. The method according to claim17, wherein m is
 2. 21. The method according to claim 17, wherein m is3.
 22. The method according to claim 1, wherein a chromogenic sialidasesubstrate compound comprising the formula of General Structure I iscontacted.
 23. The method according to claim 1, wherein a compositioncomprising a chromogenic sialidase substrate of General Structure I iscontacted.